Manoyl Oxide Research Articles

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm−1 d−1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Chemical Composition and Variability of Leaf and Berry oils from Corsican Juniperus macrocarpa

The chemical composition of five leaf oil samples and eighteen berry oil samples from Corsican Juniperus macrocarpa have been investigated by GC(RI), GC-MS and 13C NMR. The composition of berry oils was dominated by monoterpene hydrocarbons with α-pinene (56.4-78.9%) as main component followed by myrcene (2.2-11.9%). Germacrene D (4.5-103%) was the major sesquiterpene. The contents of the main components of leaf oils varied drastically from sample to sample: α-pinene (28.7-76.4%), δ3-carene (up to 17.3%), β-phellandrene (up to 12.3%), manoyl oxide (up to 8.1%). The occurrence of the unusual ( Z)-pentadec-6-en-2-one (0.1-1.2%) should be pointed out. Statistical analysis (Principal Component Analysis and k- means partition) suggested a unique group with atypical samples.

Diterpenoid fingerprints in pine foliage across an environmental and chemotypic matrix: Isoabienol content is a key trait differentiating chemotypes

Diterpenoids constitute an important part of oleoresin in conifer needles, but the environmental and genetic controls on diterpenoid composition are poorly known. We studied the presence of diterpenoids in four pine populations spanning an extensive range of nitrogen (N) availability. In most samples, isoabienol was the main diterpenoid. Additionally, low contents of (Z)-biformene, abietadiene isomers, manoyl oxide isomers, labda-7,13,14-triene and labda-7,14-dien-13-ol were quantified in pine needles. According to the occurrence and content of diterpenoids it was possible to distinguish ‘non diterpenoid pines’, ‘high isoabienol pines’, ‘manoyl oxide – isoabienol pines’ and ‘other diterpenoid pines’. ‘Non diterpenoid pines’, ‘high isoabienol pines’ and ‘other diterpenoid pines’ were characteristic to the dry forest, yet the majority of pines (>80%) of the bog Laeva represented ‘high isoabienol pines’. ‘Manoyl oxide – isoabienol pines’ were present only in the wet sites. Additionally, orthogonal partial least-squares analysis showed, that in the bogs foliar nitrogen content per dry mass (NM) correlated to diterpenoids. Significant correlations existed between abietadienes, isoabienol and foliar NM in ‘manoyl oxide – isoabienol pines’, and chemotypic variation was also associated by population genetic distance estimated by nuclear microsatellite markers. Previously, the presence of low and high Δ-3-carene pines has been demonstrated, but the results of the current study indicate that also diterpenoids form an independent axis of chemotypic differentiation. Further studies are needed to understand whether the enhanced abundance of diterpenoids in wetter sites reflects a phenotypic or genotypic response.

Comparison of essential oils from Cistus species growing in Sardinia

Cistus genus is present in Sardinia with large populations of C. monspeliensis, C. salvifolius, C. creticus subsp. eriocephalus and few stations of C. albidus, C. creticus subsp. creticus and C. creticus subsp. corsicus. No chemical studies are currently being carried on Cistus species of Sardinia. The essential oils have shown six different profiles. C. creticus subsp. eriocephalus showed a high amount of manoyl oxide and its isomer (70%). C. salvifolius has pointed out the group of labdans, (20%); another consistent percentage is made of perfumed molecules as ionone and its derivate. Several linear hydrocarbons were produced by C. monspeliensis, and the heneicosane was the most represented element. In C. albidus no labdane-type diterpenes were identified. Analysis of C. creticus subsp creticus revealed several oxygenated sesquiterpenes and labdane-type diterpenes, especially manoyl oxide. C. creticus subsp. corsicus was qualitatively very similar to C. creticus subsp. creticus, notably concerning the labdane-type compounds.

Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.

BackgroundSeveral plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast.ResultsSince the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L−1.ConclusionsIn this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0440-8) contains supplementary material, which is available to authorized users.

Essential oil composition and enantiomeric distribution of some monoterpenoid components of Juniperus communis L. from Algeria

The chemical composition of essential oil isolated from the needles of Juniperus commuis L. (Cupressaceae) by hydrodistillation was analyzed by capillary GC-FID and GC/MS. A total of seventy-four compounds representing 93.35% of the oil were identified, the main components from oil were α-pinene (14.2%) followed by sabinene (12.4%), terpinene 4-ol (14.1%), γ-terpinene (5.9%), (2Z,6E) farnesol (5.4%) and manoyl oxide (4.1%). The enantiomeric distribution of α-pinene, sabinene, camphene, β-pinene, limonene, terpinen-4-ol, borneol and myrtenol were successfully determined in essential oil of J. communis.

Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G.robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the invitro and invivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G.robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Chemical composition and biological activity of the essential oil of Ballota hispanica (L.) Benth. growing wild in Sicily

Ballota L. comprises several relevant species largely used for their excellent therapeutic properties. Ballota hispanica (L.) Benth. is widely used in herbal medicine, and it is sold in herbalist shops for its sedative and antispasmodic properties. Considering its traditional medicinal use and the lack of scientific studies on the volatile components of this species as well as on its biological activities, in this study the chemical composition of the essential oil from aerial parts of B. hispanica, collected in Sicily, was evaluated by gas chromatography and gas chromatography–mass spectrometry. α-Elemol was the most abundant component of the oil (10.9%), followed by α-ylangene (8.5%), γ-dodecalactone (5.1%), and manoyl oxide (4.8%). A comparison was made of the composition of the different Ballota taxa studied so far showing a peculiar profile of B. hispanica. Futhermore, the antimicrobial and the free radical scavenging activities of the oil were determined.

Composition of the essential oils of three Cyperus species from Congo

Essential oils from underground parts of Cyperus articulatus (L.), Cyperus sp. and Kyllinga brevifolia (S.) were analyzed by gas chromatography mass spectrometry (GC-MS). Thirty four (34) components were identified in the oil of C. articulatus. The main components were α-cadinol (12.07%), trans-pinocarveol (9.86%), cyperenone (7.28%), cyperene (6.29%), myrtenol (5.50%), myrtenal (4.90%) and cis-carveol (4.42%). Fifteen (15) components were identified in the oil of Cyperus sp. The main components were farnesol (28.61%), humulene epoxide (12.86%) and caryophyllene oxide (8.96%). Twenty three components were identified in the oil of K. brevifolia with the main components being manoyl oxide (44.08%), β-pinene (13.58%), cyperene (7.63%) and γ-terpineol (7.37%). Only β-pinene and limonene were present in the three species of Cyperaceae. The findings show broad differences in chemical compositions of the essential oils of C. articulatus, Cyperus sp. and K. brevifolia. When compared with previous studies available in literature, it was also noticed that a difference of composition existed for oils of C. articulatus and K. brevifolia of other origins, respectively. Key words: Cyperus articulatus, Cyperus sp., Kyllinga brevifolia, Cyperaceae, essential oil, rhizome.

Chemical compositions and anti-inflammatory activities of essential oils from Aster spathulifolius and Vitex rotundifolia maxim

Chemical Constituents of the Essential Oil of Laggera pterodonta (DC.) Sch. Bip. From North-Central Nigeria Egharevba Henry Omoregie, Kunle Folashade Oluyemisi, Okwute Simon Koma, Okogun Joseph Ibumeh

Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form.

Forskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13R)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals.

Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M.vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the invitro and invivo functional characterization of the M.vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual βα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E.coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

Vasorelaxant effects in aortic rings of eight diterpenoids isolated from three Venezuelan plants

Vasorelaxant effects of eight diterpenoids isolated from three Venezuelan plants [(+)-manool [(+)-labda-8(17),14-dien-13-ol], (+)-manoyl oxide, (+)-2-oxomanoyl oxide, sandaracopimara-8(14), 15-dien-3β, 19-diol, jhanidiol acetate (18-acetoxy-1β- hydroxymanoyl oxide), jhanidiol (1β,18-dihydroxymanoyl oxide), ent-kaur-16-en-19- ol and grandiflorenic acid (ent-kaur-9(11),16-dien-19-oic acid)] aortic rings were assessed in intact endothelium and endothelium-denuded isolated rat. Thw cumulative addition (10−6 to 10−4 M) of each product were carried out after contraction with phenylephrine (10−6 M). Jhanidiol acetate and ent-kaur-9,16-en-19-oic acid at 10−4 M dose concentration, exhibit the maximal vasorelaxant effect in endothelium-intact rings (51.61±7.62% and 79.27±7.41%, respectively). In endothelium-denuded aortic rings, the maximum vascular response exerted by both compounds was not abolished (64.14±5.64% and 84.84±3.62%, respectively). In denuded aortic rings, the half-maximal inhibitory concentration (IC50) Jhanidiol was obtained by the ethyl less than those obtained in rings endothelium (1.09×10−4 vs 7.29×10−5 M, respectively), although this difference was not significant. These results suggested that the mechanism behind the vasorelaxant effect of the two diterpene is mediated by endothelium-independent pathways.

Chemical composition of the terpenoids in wood and knots of Abies species

Oily constituents were obtained from sapwood and knots of Abies balsamea and A. alba using steam-distillation, and the chemical compositions were determined by gas chromatography and gas chromatography–mass spectrometry. The oily constituents of sapwood and knots of A. balsamea were mainly oxygenated diterpenes with manoyl oxide (1), epi-13-manoyl oxide (2), manool (3), and epi-13-manool (4). Moreover, 2 and 3 were found to be components in A. alba. The constituents of sound knots were dominated by oxygenated diterpenes, together with 2 and 3. Comparison of the content of oxygenated diterpenes from the material revealed that dead knots of A. balsamea contained a large quantity of 4, which was higher than in sapwood. Knots of both Abies spp. contained large quantities of oxygenated diterpenes that were also higher than in sapwood.

Extraction of essential oil from Cupressus sempervirens: comparison of global yields, chemical composition and antioxidant activity obtained by hydrodistillation and supercritical extraction†

In this study, supercritical fluid extraction (SFE) with CO2 and hydrodistillation (HD) were compared as methods to isolate the essential oil from Cupressus sempervirens. The odour of the oil obtained by SFE at 90 bar and 40°C was very close to the odour of the leaves of C. sempervirens before the extraction. Compounds extracted by both SFE and HD were identified by GC-FID and GC-MS. Moreover, the difference in the chemical composition obtained by SFE and HD was quite noticeable qualitatively and quantitatively. Phenolic composition and antioxidant activity were also determined. Compared to HD, the SFE method presents some advantages: the extraction was completed after 1 h in SFE, although 4 h is necessary for HD, and the yield was improved by 34%. Finally, it has also been shown that SFE is very selective towards some specific components such as manoyl oxide, trans-totarol and α-acoradiene.

A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate

The bicyclic diterpene (-)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (-)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 [M+H](-)). The enzymatic reaction was also carried out in the presence of 60% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases.

Identification of volatiles from Pinus silvestris attractive for Monochamus galloprovincialis using a SPME-GC/MS platform

IntroductionA myriad of volatile organic compounds (VOCs) released by terrestrial vegetation plays an important role in environmental sciences. A thorough chemical identification of these species at the molecular level is essential in various fields, ranging from atmospheric chemistry to ecology of forest ecosystems. In particular, the recognition of VOCs profiles in a context of plant–insect communication is a key issue for the development of forest protection tools.PurposeThis work was aimed at the development of a simple, robust and reliable method for the identification of volatiles emitted from plant materials, which can attract or deter pest insects. Specifically, volatiles emitted from the bark of Pinus sylvestris were studied, which might attract the black pine sawyer beetle Monochamus galloprovincialis—a serious pest of the tree and a vector of a parasitic nematode Bursaphelenchus xylophius. MethodThe volatiles from bark samples were collected using a solid-phase micro-extraction technique, and subsequently analysed by gas-chromatography/mass-spectrometry (GC/MS). The characterisation of the volatile fraction was based on the comparison with data in mass spectral libraries, and in most cases, with the available authentic standards. The identified compounds were screened against the available entomological data to select insect attractors.ResultsThe identified components included terpenes (α-pinene, ∆-3-carene, and para-cymenene), oxygenated terpenes (α-terpineol and verbenone), sesquiterpenes (α-longipinene, longifolene, E-β-farnesene, γ-cadinene and pentadecane), and diterpenes (manoyl oxide and (+)-pimaral). Of these, longifolene and (+)-pimaral are of particular interest as plausible attractors for the M. galloprovincialis beetle that might find application in the construction of insect bait traps.

Essential Oil Variability in Natural Hahadjerine Population of Cupressus dupreziana in Tassili Ajjer (Algeria)

Essential oils extracted from dried leaves of Cupressus dupreziana A. Camus, an endemic species in the Tassili n’Ajjer (Sahara Central Algeria), were analysed by gas chomatography coupled to mass spectrometry (GC-MS). Terpinoid analyses were performed on 13 trees in the natural population of Hahadjerine in order to determine the intra-population variability. 39 trepenoids were identified; the averages of the principal components were trans- totarol (24.4%), Manoyl oxide (21.2%), α-pinene (15%) and Δ3-carene (11.3%). The terpenoid markers used made it possible to determine the individual patterns of chemotypic variability. This variability confirms that genetic factors are not solely responsible for the decrease in the numbers of this species.

Leaf Essential Oil Composition of Kyllinga brevifolia Rottb. from Nepal

The essential oil from the leaf of Kyllinga brevifolia Rottb., collected from Biratnagar, Nepal, was obtained by hydrodistillation and analyzed by GC-MS. A total of 22 compounds were identified in the oil accounting for 96.1 % of the oil composition. The majority of the essential oil was identified to be occadinol (40.3 %),’L-τ-muurolol (19.5 %), and germacrene D-4-ol (12.5 %). The oil also contained smaller amounts of δ-cadinene (2.9 %), and germacrene D (4.0 %). Reports on the essential oil composition of K. brevifolia from Nepal were compared with a sample from Brazil, which showed that 13-epi-manoyl oxide (26.1 %) and manoyl oxide derivatives constituted the majority of composition with a-cadinol comprising only 0.4 %. However germacrene D was found in similar percentages between Nepal and Brazil samples (4.0 %and 4.2 %, respectively).